Daurichromenic acid and grifolic acid: Phytotoxic meroterpenoids that induce cell death in cell culture of their producer Rhododendron dauricum.

Daurichromenic acid (DCA) is a meroterpenoid with anti-HIV activities that is isolated from Rhododendron dauricum L. We recently reported that DCA is biosynthesized and accumulated in the apoplast of glandular scales attached on the surface of young leaves of R. dauricum. In the present study, we confirmed that a cell suspension culture of R. dauricum could not produce DCA and its precursor grifolic acid even after elicitation with methyl jasmonate and ß-cyclodextrin. In addition, exogenous supplementation of DCA and grifolic acid effectively induced cell death in the same culture, with apoptosis-associated phenomena such as cytoplasmic shrinkage, chromatin condensation, and genomic DNA degradation. These findings suggested that DCA and grifolic acid are phytotoxic metabolites that have to be sequestered in the apoplast to avoid self-poisoning.

Identification and Characterization of Daurichromenic Acid Synthase Active in Anti-HIV Biosynthesis.

Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from Rhododendron dauricum We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of R. dauricum The gene coded for a 533-amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino-terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine-112 and cysteine-175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic Pichia pastoris, exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from Cannabis sativa, whereas DCA synthase catalyzes a novel cyclization reaction of the farnesyl moiety of a meroterpenoid natural product of plant origin. Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of R. dauricum plants, based on various analytical studies at the chemical, biochemical, and molecular levels. The extracellular localization of DCA also was confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA: the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells.

Is the root-colonizing endophyte Acremonium strictum an ericoid mycorrhizal fungus?

In previous investigations, we found that Acremonium strictum (strain DSM 100709) developed intracellular structures with similarity to mycelia of ericoid mycorrhizal fungi in the rhizodermal cells of flax plants and in hair roots of Rhododendron plantlets. A. strictum had also been isolated from roots of ericaceous salal plants and was described as an unusual ericoid mycorrhizal fungus (ERMF). As its mycorrhizal traits were doubted, we revised the hypothesis of a mycorrhizal nature of A. strictum. A successful synthesis of mycorrhiza in hair roots of inoculated ericaceous plants was a first step of evidence, followed by fluorescence microscopy with FUN(®)1 cell stain to observe the vitality of the host cells at the early infection stage. In inoculation trials with in vitro-raised mycorrhiza-free Rhododendron plants in axenic liquid culture and in greenhouse substrate culture, A. strictum was never observed in living hair root cells. As compared to the ERMF Oidiodendron maius and Rhizoscyphus ericae that invaded metabolically active host cells and established a symbiotic unit, A. strictum was only found in cells that were dead or in the process of dying and in the apoplast. In conclusion, A. strictum does not behave like a common ERMF-if it is one at all. A comparison of A. strictum isolates from ericaceous and non-ericaceous hosts could reveal further identity details to generalize or specify our findings on the symbiotic nature of A. strictum. At least, the staining method enables to discern between true mycorrhizal and other root endophytes-a tool for further studies.

Alp Rose stem cells, olive oil squalene and a natural alkyl polyglucoside emulsifier: Are they appropriate ingredients of skin moisturizers - in vivo efficacy on normal and sodium lauryl sulfate - irritated skin?.

Background/Aim: Since skin moisturization may be achieved by both actives and chosen carrier, plant stem cells, squalene and natural alkyl polyglucoside emulsifier may be potential components of contemporary cosmetic products. The aim of the study was in vivo evaluation of the skin irritation potential and the efficacy of Alpine Rose stem cells incorporated into li-posomes and olive oil squalene as ingredients of moisturizing creams, with respect to the novel emulsifier used for creams' stabilization. Methods: With the employment of noninvasive skin biophysical measurements, skin hydration (EC), transepi-dermal water loss (TEWL), erythema index (EI) and viscoelas-ticity were measured on 76 healthy volunteers. In the first phase, skin irritation after a 24-hour occlusion and the long-term efficacy of creams (a 21-day study) on healthy skin were evaluated. Phase II of the study focused on the cream efficacy assessment after a 6-day treatment of sodium lauryl sulfate-irritated skin. Results: After a 24-hour occlusion, there were no significant changes in the EI for any tested sample. In the second phase of the study, the EI was not significantly altered for the cream containing squalene, while the application of all active samples resulted in a significant reduction of TEWL. In both phases of the study an EC increase was recorded, espe-cially for the squalene-containing cream. Conclusion: Due to the lack of skin irritation and skin barrier impairment along with the marked hydration effect, it could be said that the in-vestigated actives incorporated into alkyl polyglucoside emulsi-fier-stabilized creams may be safely applied as ingredients for "tailor-made" cosmetic moisturizers intended for normal and dry skin care, whereas olive oil squalene could be used for the treatment of irritated or sensitive skin as well. [Projekat Ministarstva nauke Republike Srbije, br. TR34031]

Leaf life span optimizes annual biomass production rather than plant photosynthetic capacity in an evergreen shrub.

SUMMARY: *Owing to nitrogen (N) translocation towards new leaves, the shedding of old leaves can increase the whole-plant carbon gain. It occurs when their photosynthetic nitrogen use efficiency (PNUE) declines below a given threshold. *Here, we investigated variations in net photosynthetic capacity (A(max)), N resorption and PNUE in populations of Rhododendron ferrugineum presenting different mean leaf life spans (LLS). *Both populations had comparable annual leaf surface area production and A(max) across leaf-age cohorts. Branch photosynthetic capacity was up to 95% higher in the population with the longer LLS mainly because of the high contribution of old leaves to the total leaf area. Despite lower N concentrations, old leaves maintained relatively high A(max) and consequently PNUE that were higher than or similar to the values found in current-year leaves. *As the ratio of PNUE in old to PNUE in new leaves was always higher than the fraction of leaf N resorbed during leaf shedding, we concluded that leaf shedding did not improve plant photosynthetic capacity. We suggest that in R. ferrugineum, leaf shedding is mainly controlled by the leaf storage function and, therefore, that models aiming to explain LLS should not only consider the leaf carbon assimilation function, particularly in nutrient-poor habitats.

Authentication of the 31 species of toxic and potent Chinese Materia Medica by light microscopy, Part 3: two species of T/PCMM from flowers and their common adulterants.

Toxic and potent Chinese Materia Medica (T/PCMM) has become a hot and sensitive topic as more and more people around the world are interested in the safety of herbal medicines. T/PCMM is irreplaceable in treating some diseases; but it can easily cause serious problems if confused with other herbal medicines. Accurate identification is essential to ensure their safe use, but up to now, the literature on the authentication of T/PCMM is scant. Thus, we are undertaking a study of 31 T/PCMM originating from plants, animals, minerals, and secreta. Our previous study established microscopic observation as a simple, fast, accurate, and convenient method for identifying and authenticating animal and seed T/PCMM. This study focused on the authentication of flower T/PCMM as a part of the whole study. The flower T/PCMM studies were derived from two species, Datura metel L. (Flos Daturae) and Rhododendron molle G. Don (Flos Rhododendri Mollis). Other species easily confused with these two were also examined and characterized. Using the microscope camera, normal light and polarized light microscopy, we determined the macroscopic and microscopic features of the flowers; in addition, the oil immersion lens was used to study the pollen grain characteristics. The results demonstrated that flower T/PCMM can be identified and authenticated using a light microscope equipped with an oil immersion lens. This same equipment can be easily used to characterize other herbal flower medicines.

Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence.

Infrared differential thermal analysis (IDTA) and differential imaging chlorophyll fluorescence (DIF) were employed simultaneously to study the two-dimensional pattern of ice propagation in leaves and mesophyll freeze dehydration as detected by a significant increase of basic chlorophyll fluorescence (F(0)). IDTA and DIF technique gave different insights into the freezing process of leaves that was highly species-specific. IDTA clearly visualized the freezing process consisting of an initial fast spread of ice throughout the vascular system followed by mesophyll freezing. While mesophyll freezing was homogeneously in Poa alpina, Rhododendron ferrugineum and Senecio incanus as determined by IDTA, DIF showed a distinct pattern only in S. incanus, with the leaf tips being affected earlier. In Cinnamomum camphora, a mottled freezing pattern of small mesophyll compartments was observed by both methods. In IDTA images, a random pattern predominated, while in DIF images, compartments closer to lower order veins were affected earlier. The increase of F(0) following mesophyll freezing started after a species-specific time lag of up to 26 min. The start of the F(0) increase and its slope were significantly enhanced at lower temperatures, which suggest a higher strain on mesophyll protoplasts when freezing occurs at lower temperatures.

[Pharmacognostic studies of Tibetan medicine da-li].

Da-Li is a traditional medicine of Tibetan, its original plant is Rhododendron primulaeflorum Bur. et Franch and R. anthopogonoides Maxim. This paper reports the identification of Flos et Folium Rhododendri Primulaeflori on its macroscopic character, microscopical charactersitic and TLC. The comparison between the Flos et Folium Rhododendri Primulaeflori and Flos et Folium Rhododendri anthopogonoidi is also reported.